GAG protein is the major constituent of the HIV-1 virion core, and it interacts with viral RNA in a number of ways that are critical for the viral lifecycle. Specific Gag binding to the 5' packaging (or psi) region in the HIV-1 genome is essential for targeting this RNA into nascent viral particles, and helps initiate formation of the genomic dimer. Gag/RNA interactions also promote tRNA priming, reverse transcription, virion assembly, and other crucial processes, and so offer an especially promising target for new antiviral therapies. Our goal is to dissect the various molecular interactions of Gag with HIV-1 RNA in vitro, and to evaluate their functions genetically in the live virus. We have previously characterized sites of Gag-binding and RNA dimer contact in the psi locus. We now propose to explore the functional interactions among those sites in detail, to map additional sites believed to lie upstream, and to search for others elsewhere in the genome that may contribute to virion assembly and maturation. We will attempt to delineate all of the cis-acting signals required for HIV-1 packaging in vivo. Our earlier studies revealed a new class of HIV-1 mutants that have a selective defect in RNA dimerization, associated with markedly reduced infectivity. By analyzing those mutants, we will evaluate the precise contributions of genomic dimerization to packaging, reverse transcription, sequence recombination, RNA stability, and the conformation of the genome within the HIV-1 core. Comparative studies in murine leukemia virus and the human telomerase complex are planned. We have also identified a heterologous RNA ligand that binds HIV-1 Gag with high affinity and has packaging activity in vivo; we now propose to investigate whether this ligand can act as an effective competitive inhibitor of Gag/psi binding and of HIV-1 replication. Together, these studies will illuminate particular Gag/RNA interactions that are vulnerable drug targets, and will provide novel in vitro assays for exploiting those targets in antiviral drug discovery.